Bioactive peptides from natural sources

Nature`s diversity has always been and still is one of the biggest resources of therapeutic lead compounds. Many natural products exhibit biological activity against unrelated targets, thus providing researchers with starting points for drug development. Natural peptides of great number and diversity occur in all organisms from microbes to plants to man. Natural peptide libraries offer a unique way to screen the diversity of peptide-protein interactions for the modification of G protein-coupled receptor (GPCR) signalling response and drug lead discovery. GPCRs are considered to represent the most promising drug targets and 30-50% of the currently marketed drugs elicit their actions by binding to GPCRs, although only ~60 GPCRs are exploited as drug targets. Natural peptide libraries are likely to play a major role in identifying new GPCR ligands. In particular venom peptides from marine cone-snails (conotoxins) and circular peptides from plants (cyclotides) have been established as a rich source of drug leads. The reported uterotonic activity and neurotensin antagonism of cyclotides are of particular interest as these are the first examples of cyclotides possibly targeting GPCRs. The mechanisms of how these activities are brought about are still not known. Hence it is intriguing to further explore these effects, especially since some cyclotides contain sequence- and structural-motifs that are similar to the presumed active motifs in the human oxytocin (OT) peptide. The recent discovery and characterization of conopressin-T in comparison with vasopressin (AVP) and OT led to the identification of an agonist/antagonist switch, which has the propensity to be investigated further regarding novel ligand design for the human receptors. The proposed project involves the (i.) isolation and analytical characterization of naturally-occurring plant peptides, (ii.) the pharmacological characterization of plant cyclotides and conopressin-T peptide analogues and (iii.) mechanistic study of the mode-of-action of plant cyclotides. The overall aim is to identify and characterize novel peptide drug candidates, with primary focus on the OT-/ AVP-receptors. If successful, these peptide drug candidates are amenable for future synthetic optimization using ligand-based design and may well be suitable for clinical development. At the very least, we will establish a rapid GPCR screening platform for pharmacological testing of naturally-occurring and synthetic compound libraries. Furthermore, the project will lead to novel insights into the diversity and biological activity of plant cyclotides and other bioactive peptides. The development of rapid and high-resolving screening techniques of plant extracts offers the possibility for future plant peptidomics projects that trigger the discovery of novel plant cyclotides and related peptides.

Natural peptides of great number and diversity occur in all organisms, but their biological role is often unknown. Based on the original discovery of circular peptides, called cyclotides, in an African plant Oldenlandia affinis and their use in traditional medicine to accelerate labour, we studied the mechanisms underlying their biological activities which previously were unknown; specifically, a receptor for a native cyclotide had not been reported. Using bioactivity-guided fractionation of a herbal peptide extract known to indigenous healers as kalata-kalata the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a-receptors, members of the G protein-coupled receptor (GPCR) family, as molecular targets for this cyclotide. Furthermore we showed that cyclotides can serve as templates for the design of selective GPCR ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. The observations provide a proof-of-concept for the development of cyclotide-based peptide ligands. This has great impact since GPCRs are promising drug targets: >30% of the currently marketed drugs elicit their actions by binding to these transmembrane receptors. However, only ~10% of all GPCRs are targeted by approved drugs. Resorting to plant-derived compounds catalogued by ethnopharmacological analyses may increase this repertoire. The discovery of an oxytocin-like nonapeptide with enhanced receptor selectivity highlighted the potential of cyclotides for the discovery of peptide-based GPCR ligands.